Project Summary: Signal transduction systems in bacteria provide the molecular basis for coupling environmental signals to appropriate adaptive responses. One of the most prevalent signaling strategies in bacteria is a phosphotransfer pathway between two-conserved proteins, a histidine protein kinase and a response regulator. These pathways, termed two-component systems, are widespread, with >9000 systems identified in -300 sequenced bacterial genomes to date. This project focuses on characterization of response regulators, proteins which function as phosphorylation-activated switches to control output responses of the systems. The OmpR/PhoB subfamily of response regulators, distinguished by a winged- helix DMA-binding domain, accounts for -one third of all response regulators and -half of all response regulator transcription factors. It has been recently established that OmpR/PhoB response regulators in their inactive states display different arrangements of their homologous domains, but upon phosphorylation adopt a common dimeric active state mediated by a conserved molecular surface. A primary aim of this project is to measure affinities for homo- and heterodimerization of OmpR/PhoB proteins using FRET to monitor interactions in vitro and in vivo to determine whether the common active state allows heterodimerization, providing a mechanism for integrating different two-component systems within a single cell. A second aim is to determine mechanisms through which different domain arrangements in inactive OmpR/PhoB proteins regulate their transition to an active state. A third aim is to characterize the complexity of transcriptional regulation by E. coli OmpR/PhoB response regulators on a genomic scale using a combination of structural, ChlP-on-chip, and bioinformatics analyses. Additional studies will focus on structural and functional characterization of protein-DNA interactions of OmpR/PhoB and LytTR response regulators. Relevance: In addition to their importance for basic competitiveness in natural environments, two- component signaling systems are often essential for virulence when pathogenic bacteria (e.g. Mycobacterium tuberculosis, Staphylococcus aureus, Salmonella enterica) infect their hosts. Hence, understanding the molecular details of signaling pathways and their protein components provides a foundation for the development of antimicrobial drugs.